Up-regulation of activation-induced cytidine deaminase and its strong expression in extra-germinal centres in IgG4-related disease

Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a systemic disorder involving benign mass formation due to fibrosis and intense lymphoplasmacytosis; the chronic inflammation associated with the disease might also contribute to oncogenesis. Activation-induced cytidine deaminase (AID), normally expressed in germinal centre activated B-cells, is an enzyme that edits DNA/RNA and induces somatic hypermutation and Ig class switching. AID expression is strictly controlled under physiological conditions; however, chronic inflammation and some infectious agents induce its up-regulation. AID is overexpressed in various cancers and may be important in chronic inflammation-associated oncogenesis. We examined AID expression in IgG4-related sialadenitis (n = 14), sialolithiasis (non-specific inflammation, n = 13), and normal submandibular glands (n = 13) using immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Immunohistochemistry revealed significantly more AID-expressing cells in IgG4-related sialadenitis than in sialolithiasis or normal submandibular gland samples (P = 0.02 and P < 0.01, respectively); qPCR yielded similar results. Thus, AID was significantly more up-regulated and had higher expression in extra-germinal centres in IgG4-RD than in non-specific inflammation or normal conditions. This report suggests that IgG4-RD has several specific causes of AID up-regulation in addition to inflammation. Furthermore, chronic inflammation-associated AID-mediated oncogenesis is possible in IgG4-RD.

SCientifiC RepORTS | (2019) 9:761 | DOI: 10.1038/s41598-018-37404-x variable region and gives rise to mutations in the constant region to cause Ig class switching from IgM to IgG, IgA, or IgE. Under normal conditions, AID expression is strictly regulated to prevent unfavourable somatic mutations. However, there have been many reports of inappropriate AID expression in various cancers 9,10 . In addition to B-cells, AID expression can be induced in various epithelial tissues due to chronic inflammation and infection 11 . Up-regulation of AID during inflammatory conditions is believed to cause uncontrolled somatic mutations of various genes, resulting in chronic inflammation-associated cancers 12 .
The mechanism underlying oncogenesis in IgG4-RD remains unclear, but AID up-regulation due to chronic inflammation could be a contributing factor. Despite this, AID expression in IgG4-RD has not been previously examined. Therefore, in this study, we investigated the expression of AID in IgG4-related sialadenitis.
Quantitative analysis of AID expression. Quantitative real-time polymerase chain reaction (qPCR) revealed that AID expression in IgG4-RD was significantly higher than that in sialolithiasis (P = 0.02) or normal submandibular gland (P < 0.01) (Fig. 3). AID expression was also significantly higher in sialolithiasis than in normal control tissues (P < 0.01).

Discussion
The aetiologies of IgG4-RD and its underlying oncogenic mechanisms remain largely unknown. Some studies have failed to find any relationship between IgG4-RD and cancer 13,14 , whereas others have shown malignancies develop in a subset of patients with IgG4-RD 15 . Hence, the relationship between the two remains controversial. However, the frequency of KRAS mutations has been reported to be higher in the pancreatic and bile ducts and gastrointestinal tracts of patients with autoimmune pancreatitis 16,17 . This indicates that IgG4-RD is associated with somatic oncogene mutations and may induce malignancies in various organs. The development of MALT lymphomas in patients with IgG4-RD has also been documented 6,7 . Interestingly, unlike conventional MALT lymphomas, those arising in patients with IgG4-RD are associated with up-regulation of Th2 and Treg cytokines 18 . Thus, these cytokines may be important in the pathogenesis of IgG4-RD; hence, MALT lymphoma may develop due to Th2/Treg-predominated chronic inflammation. Various cancers occur in association with chronic inflammation. Infectious agents such as Helicobacter pylori and hepatitis B and C viruses are known to induce cancers related to chronic inflammation. Additionally, several non-infectious inflammatory conditions, such as inflammatory bowel disease, primary sclerosing cholangitis, and chronic pancreatitis, are also associated with malignancies 12 . The accumulation of genetic and epigenetic alterations, caused by chronic inflammation, is thought to be important in inflammation-associated oncogenesis. The majority of nucleotide alterations in cancers, including inflammation-associated cancers, involve cytosine (C)/guanine (G) to thymine (T)/adenine (A) transitions 19 . AID deaminates C residues to uracil (U), resulting in U/G mismatches. Without DNA repair, each U/G pair is replicated as T/A instead of C/G, representing the most frequent nucleotide transition observed in cancers. U/G mismatches can also be recognized by uracil-DNA glycosylase, which induces various mutations at such sites. In addition, the mutS homolog 2/mutS homolog 6 heterodimer can induce nucleotide mutations at both U/G and A/T sites near U/G mismatches. Hence, AID can induce several nucleotide sequence changes. AID contributes to somatic hypermutation and Ig class switching under physiological conditions 20 . However, AID is also known to contribute to oncogenesis by inducing unfavourable somatic mutations and even chromosomal translocations. In hematopoietic malignancies, B-cell lymphomas with up-regulated AID have been shown to carry adverse mutations in genes, such as MYC, PIM1, and PAX5, as well as chromosomal abnormalities, including MYC/IGH rearrangements 12,21-23 . Aberrant AID expression in non-lymphoid cells has also been implicated in the development of malignancies 24 , with its up-regulation inducing somatic mutations in genes, such as TP53, CDKN2A, and MYC [24][25][26][27] . A previous report also described the relationship between AID expression and IgH rearrangements in the peripheral blood of patients with Mikulicz disease 28 .
In this study, AID was shown to be up-regulated in IgG4-related sialadenitis. AID expression was also elevated in sialolithiasis, but to a significantly lesser extent. These results indicate the existence of a mechanism other than inflammation leading to elevated AID levels in patients with IgG4-RD. Under physiological conditions, AID is up-regulated by the nuclear factor-kappa B (NF-κB) signalling pathway in a T-cell-dependent or -independent manner 29 . The NF-κB pathway plays a pivotal role in oncogenesis by inducing tumour cell proliferation and suppressing apoptosis. Infectious agents, such as hepatitis C virus and H. pylori, can induce NF-κB signalling to up-regulate AID 24,30 . Further, the NF-κB-dependent induction of proinflammatory cytokine expression, including that of tumour necrosis factor, is associated with AID overexpression 11 . In addition to these proinflammatory cytokines, the Th2/Treg cytokines IL-4, IL-13, and TGF-β are also known to result in elevated AID levels 12,28,31 . Further, Treg cytokines (IL-10 and TGFβ) and AID expression were significantly higher in the labial salivary glands of patients with IgG4-RD than in healthy controls or in those with Sjögren syndrome 32 . Hence, Th2/ Treg-mediated immunity in IgG4-RD may be responsible for the observed up-regulation of AID.
Given the existence of germinal centre hyperplasia in IgG4-related sialadenitis, AID up-regulation may partially reflect increased numbers of germinal centres. However, histopathological examinations revealed many AID-positive lymphoid cells, plasma cells, and plasmacytoid cells in both the interfollicular areas and germinal centres of submandibular gland specimens from patients with sialadenitis, indicating broader aberrant AID expression. Thus, the Th2/Treg cytokines produced by interfollicular T-cells or other immune cells may cause such aberrant up-regulation.

Conclusion
This study represents the first report of AID up-regulation and strong expression in extra-germinal centres in IgG4-RD. Thus, in this disease, AID may be up-regulated by Th2/Treg-predominated immunity and induce unfavourable somatic mutations in various organs. As a result, AID may play an important role in oncogenesis in patients with IgG4-RD.

Samples.
Formalin-fixed paraffin-embedded tissues from patients with IgG4-related sialadenitis (14 cases), sialolithiasis (non-specific inflammation, 13 cases), and normal submandibular glands (13 cases) were examined. The median patient ages were 67.5 (range, 60-82), 42 , and 75 (49-92) years, respectively; the male/female ratios were 12/2, 8/5, and 12/1, respectively. This study was approved by the institutional ethical review board of Okayama University. Because the samples were limited to excess human tissue, the ethical review board waived the need for written consent from the patients.
Confirmation of histological IgG4-RD diagnosis. All cases were reviewed by two pathologists. In accordance with the consensus statement for the pathological assessment of IgG4-RD, different high-power fields (eyepiece, 10×; lens, 40×) were examined to calculate the average number of IgG4-positive cells per field (>100) and the IgG4-/IgG-positive cell ratio (>40%) 33 . All 14 cases were histologically consistent with IgG4-related sialadenitis.
Histological evaluation of AID expression. AID positivity was evaluated based on an intensity index, with scores of 0 (negative), 1+ (weakly positive), 2+ (moderately positive), and 3+ (strongly positive) (Fig. 4). Each specimen was evaluated by two pathologists. Statistical analysis. To compare AID expression among submandibular gland specimens from normal patients and those with IgG4-related sialadenitis or sialolithiasis, the Mann-Whitney U -test was applied (SPSS, version 24; IBM, Armonk, NY, USA). A P-value < 0.05 was considered statistically significant.